Background and Specific Aims:[unreadable] The presence of ubiquitinated protein aggregates in the brain is the histological hallmark of many neurodegenerative diseases, including Alzheimers disease and ALS. In 2006, Neumann and colleagues identified that TDP43 was the major component of the ubiquitinated protein aggregates in ALS and a form of earlier dementia. Intriguingly, TDP43 aggregation is not found SOD1-related ALS, nor in the transgenic mice expressing ALS-related SOD1G93A or G37R or G85R mutations. The understanding the reason for this lack of TDP43 deposition in SOD1-related ALS would likely give us new insight into the pathogenesis of both SOD1 and non-SOD1-related ALS. [unreadable] [unreadable] TDP-43 is initially identified as a cellular protein that binds specifically to pyrimidine-rich motifs in HIV-1 double-stranded TAR DNA, which represses transcription from the HIV-1 LTR promoter in both the presence and absence of HIV-1 Tat. It may function as a transcriptional repressor and as an activator of exon skipping, or in other roles, such as in mRNA biogenesis, apoptosis, and cell division. TDP43 is ubiquitously expressed and normally restricted to the nucleus. However, mutant TDP43 is redistributed within the neuron and sequestered as ubiquitinated and hyperphosphorylated insoluble aggregates in the perikarya and neurites in the brain and spinal cord of ALS patients. It is likely that the perturbation of TDP43 trafficking between the nucleus and cytoplasm causes the detrimental effect of mutant TDP43 to the motor neuron. However, the exact mechanism of how the mutations in TDP43 contribute to motor neuron degeneration is unclear. In addition, it remains a key issue in current ALS research to create new models of ALS that are informative and representative for the human condition, in order to investigate the biomarkers of disease, understand the cellular interactions and potentially provide better models for drug trials. Therefore, our specific aims are:[unreadable] Aim 1: To generate and characterize TDP43 knockout and M337V knock-in mice.[unreadable] Aim 2: To investigate the molecular pathways causing the motor neuron degeneration by which mutant TDP43 may exert its possible toxic gain of function.[unreadable] [unreadable] Progress Report and Future Directions:[unreadable] To develop a hybrid of TDP43 M337V knock-in and conditional knockout mice, we have isolated a genomic DNA fragment carrying the entire TDP43 gene from a mouse genomic DNA BAC library. We will introduce the same missense mutation in the exon 5 of mouse TDP43 gene as happened in the human case, resulting in the substitution from Methionine to Valine in the residue 337. We will then flank the exons 4 and 5 with LoxP sites. Removal of this exon by Cre recombinase will generate a premature stop codon and create a conditional TDP43 knockout mouse model. At least two independent ES clones carrying the correct recombination of the targeting vector will be isolated and used for the blastocytic injection. The resulting chimeric mice will be used to get the F1 heterozygous TDP43 knock-in/conditional knockout mice. Currently, we are in the middle of construction of the gene targeting vector. Once we have the mice, the motor behavioral phenotypes and neuropathogenic abnormalities will be examined in these genetically modified TDP43 mice. Meanwhile, we will study the in vivo function of TDP43 protein in the KO mice. We will determine the subcellular distribution of TDP43 M337V mutation in neurons by immunohistochemistry. We will also plan to identify TDP-43 assocaied proteins by immunoprecipitation from brain tissues.